Array fabrication

ABSTRACT

A method, apparatus, and computer program, for fabricating multiple arrays arranged successively in a first direction on a substrate and each having multiple feature sets arranged successively in the first direction within the array. The method uses a head system having multiple successive sets of dispensers. In the method, the head system is advanced in the first direction over the substrate while dispensing drop sets for each array from dispenser sets so as to form the arrays. In one aspect, drop sets are dispensed in an order the reverse of that from which the dispenser sets pass over a given location on the substrate as the head system advances in the first direction. In this case, each dispenser set deposits a drop set at a distance ahead of a drop set deposited by a preceding dispenser set which is less than the distance to the successive drop dispenser set which deposits the next drop set.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/629,500 filed Jul. 31, 2000, now U.S. Pat. No. 6,613,893,from which priority is claimed under 35 U.S.C. 120 and which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to arrays, particularly polynucleotide arrayssuch as DNA arrays, which are useful in diagnostic, screening, geneexpression analysis, and other applications.

BACKGROUND OF THE INVENTION

Polynucleotide arrays (such as DNA or RNA arrays), are known and areused, for example, as diagnostic or screening tools. Such arrays includeregions of usually different sequence polynucleotides arranged in apredetermined configuration on a substrate. These regions (sometimesreferenced as “features”) are positioned at respective locations(“addresses”) on the substrate. The arrays, when exposed to a sample,will exhibit an observed binding pattern. This binding pattern can bedetected upon interrogating the array. For example all polynucleotidetargets (for example, DNA) in the sample can be labeled with a suitablelabel (such as a fluorescent compound), and the fluorescence pattern onthe array accurately observed following exposure to the sample. Assumingthat the different sequence polynucleotides were correctly deposited inaccordance with the predetermined configuration, then the observedbinding pattern will be indicative of the presence and/or concentrationof one or more polynucleotide components of the sample.

Biopolymer arrays can be fabricated by depositing previously obtainedbiopolymers (such as from synthesis or natural sources) onto asubstrate, or by in situ synthesis methods. Methods of depositingobtained biopolymers include dispensing droplets to a substrate fromdispensers such as pin or capillaries (such as described in U.S. Pat.No. 5,807,522) or such as pulse jets (such as a piezoelectric inkjethead, as described in PCT publications WO 95/25116 and WO 98/41531, andelsewhere). For in situ fabrication methods, multiple different reagentdroplets are deposited from drop dispensers at a given target locationin order to form the final feature (hence a probe of the feature issynthesized on the array stubstrate). The in situ fabrication methodsinclude those described in U.S. Pat. No. 5,449,754 for synthesizingpeptide arrays, and described in WO 98/41531 and the references citedtherein for polynucleotides. The in situ method for fabricating apolynucleotide array typically follows, at each of the multipledifferent addresses at which features are to be formed, the sameconventional iterative sequence used in forming polynucleotides fromnucleoside reagents on a support by means of known chemistry. Thisiterative sequence is as follows: (a) coupling a selected nucleosidethrough a phosphite linkage to a functionalized support in the firstiteration, or a nucleoside bound to the substrate (i.e. thenucleoside-modified substrate) in subsequent iterations; (b) optionally,but preferably, blocking unreacted hydroxyl groups on the substratebound nucleoside; (c) oxidizing the phosphite linkage of step (a) toform a phosphate linkage; and (d) removing the protecting group(“deprotection”) from the now substrate bound nucleoside coupled in step(a), to generate a reactive site for the next cycle of these steps. Thefunctionalized support (in the first cycle) or deprotected couplednucleoside (in subsequent cycles) provides a substrate bound moiety witha linking group for forming the phosphite linkage with a next nucleosideto be coupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide, in aknown manner.

The foregoing chemistry of the synthesis of polynucleotides is describedin detail, for example, in Caruthers, Science 230: 281–285, 1985;Itakura et al., Ann. Rev. Biochem. 53: 323–356; Hunkapillar et al.,Nature 310: 105–110, 1984; and in “Synthesis of OligonucleotideDerivatives in Design and Targeted Reaction of OligonucleotideDerivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat.No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S.Pat. No. 5,869,643, EP 0294196, and elsewhere

In array fabrication, the quantities of polynucleotide available,whether by deposition of previously obtained polynucleotides or by insitu synthesis, are usually very small and expensive. Additionally,sample quantities available for testing are usually also very small andit is therefore desirable to simultaneously test the same sample againsta large number of different probes on an array. These conditions requireuse of arrays with large numbers of very small, closely spaced features.It is important in such arrays that features actually be present, thatthey are put down accurately in the desired target pattern, are of thecorrect size, and that the DNA is uniformly coated within the feature.Failure to meet such quality requirements can have serious consequencesto diagnostic, screening, gene expression analysis or other purposes forwhich the array is being used.

However, in order to make arrays at a reasonable cost per array, it isalso important that large numbers of arrays be fabricated in a shorttime. When drops are dispensed to form the arrays, this typicallyinvolves dispensing drops from a number of dispensers in co-ordinationwith scanning the dispensers in some pattern over a substrate (with oneor more dispenser re-loadings, as desired). For example, drops for aportion of each array can be dispensed, the dispensers relocated, dropsfor the same portion of another array dispensed, and the processrepeated followed by re-loading of the dispensers and repeating theforegoing sequence for another portion of all the arrays. However, sucha pattern requires a large number of movements and hence a relativelylong time to complete.

It would be desirable then, to provide a means for fabricating multiplearrays on a substrate while keeping the movement pattern of the multipledispensers relatively simple.

SUMMARY OF THE INVENTION

The present invention provides in one aspect, a method of fabricatingmultiple arrays arranged successively in a first direction on asubstrate. Each such array has multiple feature sets within the arraywhich are also arranged successively in the first direction. The methoduses a head system is used which has multiple successive sets ofdispensers (for example, pulse jets such as piezoelectric orthermoelectric jets). In the method, the head system is advanced in thefirst direction over the substrate. Drop sets are dispensed fromsuccessive dispenser sets for each array in co-ordination with suchmovement, such that each drop set for multiple arrays are dispensed foreach array.

In one aspect of the method, drop sets are dispensed from dispenser setsin an order the reverse of that from which the dispenser sets pass overa given location on the substrate as the head system advances in thefirst direction. In this aspect, each dispenser set deposits a drop setat a distance ahead of (as measured in the first direction) a drop setdeposited by a preceding dispenser set which is less than the distanceto the successive drop dispenser set which deposits the next drop set.Thus, while a given dispenser set is depositing drops for one featureset of an array, the dispenser sets which will deposit drops forsuccessive feature sets of the same array have still not passed over theone feature set position of the same array (that is, they are still“behind” the given dispenser set in relation to the direction of headadvancement).

The arrays fabricated may have a distance between adjacent sets offeatures within the arrays, which is less than the distance betweenadjacent sets of dispensers. In fact, in one aspect of the presentinvention, arrays are fabricated by advancing and dispensing from thepreviously described head system, to obtain arrays with correspondingfeature set spacing (for example, first feature set spacing) betweenadjacent arrays which is less than the total spacing (as measured in thedirection of head advancement) of the dispenser sets which formed thearrays. Furthermore, the sets of features may extend in a directiontransverse to the first direction. In this case, the method mayadditionally include moving the head in the transverse direction anddispensing the drop sets in co-ordination with such transverse movementso as to deposit drops along such feature sets.

The “advancing” need not be a continuous motion, and in a particularaspect, the advancing and dispensing step may involve a number ofsub-steps. Such sub-steps may include: while the head system is in oneposition in the first direction, depositing drop sets from at least one,and preferably multiple different dispenser sets for feature sets ofdifferent positions within multiple arrays; advancing the head system inthe first direction to a next position; and repeating the foregoing twosteps for successive feature sets within the arrays. During therepetitions, for each of the successive feature sets within the arrays,a corresponding dispenser set is used which deposited at a same featureset position of a previous array in step the first step during aprevious cycle. This procedure may particularly be used in the casewhere the feature sets extend in a direction transverse to the firstdirection. In this case the method can additionally include moving thehead in the transverse direction during the first step and dispensingthe drop sets in co-ordination with such transverse movement so as todeposit drops along such feature sets.

Any desired number of successively arranged arrays can be fabricated onthe same substrate by the method, each array having any desired numberof features sets (for example, at least three, at least five, at leastten arrays, or at least twenty arrays each with at least three, at leastfive, at least ten, at least twenty, or at least one hundred featuresets).

The present invention can be applied to fabricating arrays of anychemical moieties, with any number of features within each set of anarray being the same or different. The invention may, for example, beapplied to fabricating arrays of monomeric moieties or polymers, such asbiopolymers. (for example, polynucleotides or peptides). In the case ofpolymeric moieties, the drop sets may contain the polymeric moietiesthemselves (such as solutions of the polynucleotides or peptides) ormonomeric moieties (such as nucleotides or amino acids) which may bereacted in sequence (through deposition of multiple different monomercontaining moieties at each of the feature locations) to form thedesired polymeric moieties. Any of the drops within a dispensed set ofdrops may be of the same or different composition, and any of thefeatures within a feature set of the array may be the same or differentcomposition (with any number of the feature sets being of the same ordifferent composition).

Each feature set of the arrays may have multiple features arrangedsuccessively in the first direction. In this case, the dispenser setsmay also be arranged successively in the first direction, with each sethaving multiple dispensers arranged successively within the set in adirection transverse to the first direction. For example, the featuresets may be rows of features arranged successively in the firstdirection (which implies, in the case of an array, that the individualrows extend lengthwise in a direction transverse to the firstdirection). The dispenser sets may also be arranged successively in thefirst direction with each set having multiple dispensers arrangedsuccessively within the set in a second direction transverse to thefirst. For example, the dispenser sets are rows of dispensers, each ofwhich extends lengthwise in the transverse direction.

The present invention further provides an apparatus of the typedescribed above in connection with any of the methods of the presentinvention. Such an apparatus may include a head system of a type alreadydescribed above, and a transport system to advance the head system inthe first direction with respect to a substrate. A processorcommunicates with the head system and transport system. The processorcan advance the head system in the first direction over the substratewhile coordinating drop dispensing from the head system with theadvancement. In particular, the processor may dispense drop sets fromsuccessive dispenser sets for each array, such that each drop set formultiple arrays is deposited by a corresponding dispenser set whichearlier deposited at a same feature set position within a previousarray, in a manner as already described. The apparatus may optionallyadditionally include a loading station with receptacles to retainmultiple different fluids such that the dispensers can be simultaneouslybrought into contact with respective receptacles for loading thedispensers with the different fluids. Each dispenser in such anapparatus may be constructed so that it holds no more than 100 μl of afluid to be dispensed as drops (or no more than 10 μl or no more than 1μl of such fluid). By the dispenser holding a certain volume isreferenced the entire dispenser including any reservoir in continuouscommunication with the remainder of the dispenser.

The present invention further provides a computer program for use withan apparatus such as already described above, having a head system-,transport system, and processor. The computer program, when loaded intothe processor, performs the steps of a method of the present invention(by controlling the apparatus, particularly the head system andtransport system, appropriately). The computer program may becommunicated to the processor (for example, from a remote location), ormay be stored on a computer program product comprising a computerreadable storage medium.

The present invention can allow for the fabrication of multiple arrayson a substrate in a relatively efficient manner with a simple movementpattern for the dispensers.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings, in which:

FIG. 1 illustrates a substrate carrying multiple arrays, such as may befabricated by methods of the present invention;

FIG. 2 is an enlarged view of a portion of FIG. 1 showing ideal spots orfeatures;

FIG. 3 is an enlarged illustration of a portion of the substrate in FIG.2;

FIG. 4 schematically illustrates operation of an embodiment of themethod of the present invention; and

FIG. 5 is an apparatus of the present invention.

To facilitate understanding, identical reference numerals have beenused, where practical, to designate identical elements that are commonto the figures.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In the present application, unless a contrary intention appears, thefollowing terms refer to the indicated characteristics. A “biopolymer”is a polymer of one or more types of repeating units. Biopolymers aretypically found in biological systems (although they may be madesynthetically) and particularly include peptides or polynucleotides, aswell as such compounds composed of or containing amino acid analogs ornon-amino acid groups, or nucleotide analogs or non-nucleotide groups.This includes polynucleotides in which the conventional backbone hasbeen replaced with a non-naturally occurring or synthetic backbone, andnucleic acids (or synthetic or naturally occurring analogs) in which oneor more of the conventional bases has been replaced with a group(natural or synthetic) capable of participating in Watson-Crick typehydrogen bonding interactions. Polynucleotides include single ormultiple stranded configurations, where one or more of the strands mayor may not be completely aligned with another. A “nucleotide” refers toa sub-unit of a nucleic acid and has a phosphate group, a 5 carbon sugarand a nitrogen containing base, as well as functional analogs (whethersynthetic or naturally occurring) of such sub-units which in the polymerform (as a polynucleotide) can hybridize with naturally occurringpolynucleotides in a sequence specific manner analogous to that of twonaturally occurring polynucleotides. For example, a “biopolymer”includes DNA (including cDNA), RNA, oligonucleotides, and PNA and otherpolynucleotides as described in U.S. Pat. No. 5,948,902 and referencescited therein (all of which are incorporated herein by reference),regardless of the source. An “oligonucleotide” generally refers to anucleotide multimer of about 10 to 100 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having any number ofnucleotides. A “biomonomer” references a single unit, which can belinked with the same or other biomonomers to form a biopolymer (forexample, a single amino acid or nucleotide with two linking groups oneor both of which may have removable protecting groups). A “peptide” isused to refer to an amino acid multimer of any length (for example, morethan 10, 10 to 100, or more amino acid units). A biomonomer fluid orbiopolymer fluid reference a liquid containing either a biomonomer orbiopolymer, respectively (typically in solution).

A “set” or “sub-set” of any item (for example, a set of features) maycontain one or more than one of the item (for example, a dispenser setmay have one or more than one dispensers). An “array”, unless a contraryintention appears, includes any one, two or three dimensionalarrangement of addressable regions bearing a particular chemical moietyto moieties (for example, biopolymers such as polynucleotide sequences)associated with that region. An array is “addressable” in that it hasmultiple regions of different moieties (for example, differentpolynucleotide sequences) such that a region (a “feature” or “spot” ofthe array) at a particular predetermined location (an “address”) on thearray will detect a particular target or class of targets (although afeature may incidentally detect non-targets of that feature). Arrayfeatures are typically, but need not be, separated by interveningspaces. In the case of an array, the “target” will be referenced as amoiety in a mobile phase (typically fluid), to be detected by probes(“target probes”) which are bound to the substrate at the variousregions. However, either of the “target” or “target probes” may be theone which is to be evaluated by the other (thus, either one could be anunknown mixture of polynucleotides to be evaluated by binding with theother). An “array layout” refers collectively to one or morecharacteristics of the features, such as feature positioning, one ormore feature dimensions, and some indication of a moiety at a givenlocation. “Hybridizing” and “binding”, with respect to polynucleotides,are used interchangeably.

When one item is indicated as being “remote” from another, this isreferenced that the two items are at least in different buildings, andmay be at least one mile, ten miles, or at least one hundred milesapart. “Communicating” information references transmitting the datarepresenting that information as electrical signals over a suitablecommunication channel (for example, a private or public network).“Forwarding” an item refers to any means of getting that item from onelocation to the next, whether by physically transporting that item orotherwise (where that is possible) and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data.

It will also be appreciated that throughout the present application,that words such as “top”, “upper”, and “lower” are used in a relativesense only. Items (such as arrays) being “successive”, “successivelyarranged” or the like terms, in a given direction, references a thoseitems being disposed in a series one after another in that direction.However, each item of successive items need not necessarily be adjacentor neighboring another such item (although they often will be). Forexample, a given group of arrays disposed successively in a firstdirection may have other arrays interposed between them which are not ofthe referenced group. “Fluid” is used herein to reference a liquid.Reference to a singular item, includes the possibility that there areplural of the same items present. Furthermore, when one thing is“advanced”, “moved”, “moving”, “re-positioned”, “scanned”, or the like,with respect to another, this implies relative motion only such thateither thing or both might actually be moved in relation to the other.For example, when dispensers are “advanced” relative to a substrate,either one of the dispensers or substrate may actually be put intomotion by the transport system while the other is held still, or bothmay be put into motion. All patents and other cited references areincorporated into this application by reference.

Referring first to FIGS. 1–3, typically methods and apparatus of thepresent invention generate or use a contiguous planar substrate 10carrying multiple arrays 12 disposed successively across a front surface11 a of substrate 10 in a both a first direction 63 a and seconddirection 204 a (the second direction 204 a being transverse to firstdirection 63 a, that is at some angle to the first direction 63 a, mosttypically orthogonal to first direction 63 a). Arrays 12 are separatedby inter-array areas 13. A back side 11 b of substrate 10 does not carryany arrays 12. All of the arrays 12 on substrate 10 may be the same orany number or all may be different. Each array 12 can be designed fortesting against any type of sample, whether a trial sample, referencesample, a combination of them, or a known mixture of polynucleotides (inwhich latter case the arrays may be composed of features carryingunknown sequences to be evaluated). While ten arrays 12 are shown inFIG. 2 and the different embodiments described below may use substrateswith particular numbers of arrays, it will be understood that substrate10 and the embodiments to be used with it, may use any number of desiredarrays 12. Similarly, substrate 10 may be of any shape, and anyapparatus used with it adapted accordingly. Depending upon intended use,any or all of arrays 12 may be the same or different from one anotherand each will contain multiple spots or features 16 of biopolymers inthe form of polynucleotides. A typical array may contain from more thanten, more than one hundred, more than one thousand or ten thousandfeatures, or even more than from one hundred thousand features. All ofthe features 16 may be different, or some or all could be the same. Inthe case where arrays 12 are formed by the conventional in situ ordeposition of previously obtained moieties, as described above, bydepositing for each feature a droplet of reagent in each cycle such asby using a pulse jet such as an inkjet type head, interfeature areas 17(as represented by the distances 17 a or 17 b) will typically be presentwhich do not carry any polynucleotide. It will be appreciated though,that the interfeature areas 17 could be of various sizes andconfigurations. It will also be appreciated that there need not be anyspace separating arrays 12 from one another. Each feature carries apredetermined polynucleotide (which includes the possibility of mixturesof polynucleotides). As per usual, A, C, G, T represent the usualnucleotides. It will be understood that there may be a linker molecule(not shown) of any known types between the front surface 11 a and thefirst nucleotide.

As illustrated in FIG. 2 in particular, each array 12 has multiple setsof neighboring features in the form of multiple rows 15 a through 15 e(only features of three rows being visible in FIG. 2). All neighboringfeatures within a feature set have the same spacing. It will beunderstood in this application that “neighboring” feature sets are thosewhich have no other feature set positioned between them. Where a givenfeature set has closest and further feature sets to which there are nointervening feature set, only the closest feature sets are considered“neighboring” feature sets. The rows 15 a through 15 e (which arefeature sets) are also arranged successively in the first direction 63a, with each row extending lengthwise in the second direction 204 a.

For the purposes of the discussions below, it will be assumed (unlessthe contrary is indicated) that the array being fabricated is apolynucleotide array formed by the deposition of previously obtainedpolynucleotides using pulse jet deposition units. However, theapplicability of the method to arrays of other polymers or chemicalmoieties generally, whether formed by multiple cycle in situ methods ordeposition of previously obtained moieties, or using other types ofdispensers, will be understood from these discussions.

Referring to FIG. 4, operation of a method of the present invention isillustrated to fabricate multiple arrays 12 such as shown in FIGS. 1 and2. FIG. 4 is a view from above looking down (using the orientation ofFIG. 5) toward a head system 210 and substrate 10 (not shown in FIG. 4for clarity) onto which an array is to be fabricated. Head system 210has two heads 210 a and 210 b. In FIG. 4 each head 210 a and 210 b isillustrated with five parallel rows and two columns (all parallel) ofdispensers. However, as described below in connection with FIG. 5, eachhead may in practice have many more rows and columns although the numberof rows and columns have been kept low in FIGS. 4 for the purposes ofclarity. Each dispenser is illustrated by its drop dispensing outlet(the drop outlet orifice, for example, in a corresponding pulse jet)represented by a hollow circle in FIG. 4. Deposited droplets arerepresented by the differently shaded circles (solid black or with apattern). Since, as described below in connection with FIG. 5, heads 210a and 210 b are both mounted to the same head retainer 208, all dropdispensers will be moved in unison by the transport system (see FIG. 5).These drop dispensers are identified as five successive sets in the formof successive rows A, B, C, D, and E in FIG. 4, and four columns 1, 2,3, and 4. Note that these rows are both successive, and sequential in adirection opposite first direction 63 a, in that as head 210 is advancedin first direction 63 a, rows E, D, C, B, and A, in that order, pass agiven position in relation to substrate 10. In the discussion of FIG. 4any particular drop dispenser will be referenced by row number followedby column number. For example, drop dispenser A1 refers to the dispenserin row A, column 1, and drop dispenser B2 refers to the drop dispenserin row B, column 2. Drops deposited by a same one of the dispenser rows,have the same shading. Referring particularly to FIG. 4G, nine fullarrays each of 4×5 features are fabricated, these arrays being in arrayrows 14 a, 14 b, 14 c, and twelve partially completed arrays arefabricated in array rows 14 d, 14 e, 14 f, and 14 g. Array rows 14 athrough 14 g are both successive, and sequential in that order in firstdirection 63 a, as they are fabricated in that order as head system 210is advanced in first direction 63 a. Similarly, rows 15 a through 15 eare successive and sequential in the first direction. All arrays are inarray columns 9 a, 9 b, 9 c, each array having feature rows 15 a through15 e and feature columns 1 through 4.

In the method, all twenty drop dispensers in head system 210 aretypically first loaded with different moieties (such as differentpolynucleotides) although one or more of the dispensers may be loadedwith the same moieties. Head system 210 is then advanced in the seconddirection 204 a over the substrate while dispensing drop sets fromsuccessive dispenser rows for each array 12, such that each drop set formultiple arrays 12 is deposited by a corresponding dispenser set whichearlier deposited at a same feature set position within a previousarray. If only one column 9 a of multiple arrays 12, for example, was tobe fabricated, then the advancing could be essentially continuous indirection 63 a. However, since multiple columns 9 a, 9 b, 9 c of arrays12 are to be fabricated, this advancing is intermittent. In particular,this advancing and dispensing operates as follows. First, while headsystem 210 remains stationary in a same position in the first direction(that is, while head system 210 does not move in first direction 63 a),it is moved in second direction 204 a with multiple sets of drops beingdeposited by dispenser row E. That is, a drop is deposited from each ofdispensers 4, 3, 2, and 1 of dispenser set E, in that order, as each ofthose dispensers passes in turn over the positions for features 4, 3, 2,1 in row 15 a of array column 9 a. This is repeated in row 15 a of arraycolumns 9 b, 9 c as head system 210 continues to travel in the seconddirection 210 a with respect to substrate 10. Head system 210 is thenreturned to the left as viewed in FIG. 4. The result of such a “firstpass” of head system 210, is drops deposited at feature locations of afirst row 15 a for each of three arrays to be formed in a row 14 a ofarrays (see FIG. 4G). Each part of FIG. 4 shows the results of anadditional pass after return of head system 210 in a direction oppositesecond direction 204 a but before further advancement of head system 210in first direction 63 a for the next pass.

Head system 210 is then advanced in first direction 63 a with respect tosubstrate 10 to a next position, and a second pass of head system 210over substrate 10 is executed. During this second pass the same patternof deposition is repeated but with drop sets being deposited from bothof dispenser rows D and E. The results of such a second pass will be theadditional deposition of drops at feature locations of a second featurerow 15 b (which is successive to feature row 15 a) for each of the threearrays to be formed in row 14 a of arrays (deposited by row D ofdispensers), and drops deposited at feature locations of a first row 15a for the three arrays to be formed in a row 14 b of arrays (depositedby row E of dispensers). A third “pass” of head system 210 is executedas illustrated in FIG. 4C but dispensing drop sets from rows C, D, and Eof dispensers. Fourth, fifth, sixth, and seventh passes are illustratedin FIGS. 4D, 4E, 4F, and 4G, respectively. Note that by pass five (FIG.4E) all dispenser rows A through E are dispensing drop sets. On eachfollowing pass, another row of arrays is completed as the top row ofnozzles in the printhead fills in the bottom row of the array. Ninecompleted arrays of twenty features each, can be seen in FIG. 4G.Further passes can be executed as desired and limited only by the sizeof substrate 10.

The number of passes required to print N rows of complete arrays usinghead system 210 as illustrated, is N+4. The final arrays are spacedequal to the dispenser nozzle spacing plus one spacing between adjacentfeatures. Alternatively, for subsequent passes in FIGS. 4B through 4G,the head system 210 can be incremented by a spacing equal to that of thenozzle rows and the features printed to the right of (as viewed in FIG.4) drops deposited from previous passes rather than below. Further,combinations of the foregoing two methods can be used. The methodselected depends on the number of fluids to be dispensed and the totalnumber of dispensers available.

Note that during each cycle (or pass) of the steps each of multiple rowsof features are deposited by a corresponding dispenser row whichdeposited at a same feature set position of a previous array during aprevious pass. For example, in the third pass (FIG. 4C) the firstfeature row 15 a of array row 14 c was deposited by dispenser row Ewhich also deposited at the same feature row position (that is, a firstrow 15 a) of array row 14 b previously deposited during the second pass.Similarly, during the third pass the second feature row 15 b of arrayrow 14 b was deposited by dispenser row D which also deposited at thesame feature row position (that is, a second row 15 b) of array row 14 apreviously deposited during the second pass. Also, as can beparticularly seen from FIG. 4G, the distance between adjacent featurerows within any of the arrays is less than the distance between adjacentdispenser sets (that is, the rows of dispensed drops are “compressed”relative to the respective dispenser rows). Similarly, columns 1–4 ofdeposited droplets are spaced closer together than the columns 1–4 ofrespective dispensers (that is, the dispensed drop columns are“compressed” relative to the respective dispensers). This decrease indeposited drop spacing in any direction of travel of the head system, isreadily obtained with pulse jet dispensers by processor 140 correctlytiming dispenser actuation as head system 210 moves over the substrate.Such compression allows for arrays with deposited drop spacing asmeasured in any direction of head travel relative to the substrate, tobe independent of the spacing of the respective dispensers whichdeposited them.

In FIG. 4, head system 210 is not reloaded. However, it will beappreciated that when the present method is used to fabricate arraysusing the in situ method as described above (wherein solutions ofmonomers are dispensed), head system 210 can be reloaded with anotherset of monomers and the procedure repeated as often as necessary tofabricate the arrays of polymers. It will also be appreciated thatarrays with a greater number of features can be obtained. This can beaccomplished, for example, by leaving sufficient space between the fourby five arrays deposited in FIG. 4 (which can then be regarded as“sub-arrays” of respective larger arrays to be fabricated). Head system210 can then be reloaded and the procedure repeated depositing furthersub-arrays in the space between the sub-arrays deposited in a firstround. It will further be appreciated that techniques of thisapplication or the two applications entitled “Array Fabrication” byPeter Webb filed the same date as the present application and bothassigned to Agilent Technologies, Inc. Ser. Nos. 09/628,963 and10/959,639, may be combined as appropriate.

Referring to FIG. 5 an apparatus of the present invention is more fullyshown, and includes a substrate station 20 on which can be mounted asubstrate 10. Pins or similar means (not shown) can be provided onsubstrate station 20 by which to approximately align substrate 10 to anominal position thereon. Substrate station 20 can include a vacuumchuck connected to a suitable vacuum source (not shown) to retain asubstrate 10 without exerting too much pressure thereon, since substrate10 is often made of glass.

A dispensing head system 210 is retained by a head retainer 208. Headsystem 210 can be positioned at any position facing substrate 10 bymeans of a transport system. The transport system includes a carriage 62connected to a first transporter 60 controlled by processor 140 throughline 66, and a second transporter 100 controlled by processor 140through line 106. Transporter 60 and carriage 62 are used execute oneaxis positioning of station 20 (and hence mounted substrate 10) facingthe dispensing head system 210, by moving it in the direction of nominalaxis 63, while transporter 100 is used to provide adjustment of theposition of head retainer 208 in a direction of nominal axis 204 (whichincludes direction 204 a) and hence move the rows of dispensers asdescribed in connection with FIG. 4. In this manner, head system 210 canbe scanned line by line, by scanning along a line over substrate 10 inthe direction of axis 204 using transporter 100, while line by linemovement of substrate 10 in a direction of axis 63 (which includesdirection 63 a) is provided by transporter 60. Head system 210 may alsooptionally be moved in a vertical direction 202, by another suitabletransporter (not shown). However, it will be appreciated that otherscanning configurations could be used. However, it will be appreciatedthat both transporters 60 and 100, or either one of them, with suitableconstruction, could be used to perform the foregoing scanning of headsystem 210 with respect to substrate 10. Thus, when the presentapplication refers to “positioning” one element (such as head system210) in relation to another element (such as one of the stations 20 orsubstrate 10) it will be understood that any required moving can beaccomplished by moving either element or a combination of both of them.An encoder 30 communicates with processor 140 to provide data on theexact location of substrate station 20 (and hence substrate 10 ifpositioned correctly on substrate station 20), while encoder 34 providesdata on the exact location of holder 208 (and hence head system 210 ifpositioned correctly on holder 208). Any suitable encoder, such as anoptical encoder, may be used which provides data on linear position.Angular positioning of substrate station 20 is provided by a transporter120, which can rotate substrate station 20 about axis 202 under controlof processor 140. Typically, substrate station 20 (and hence a mountedsubstrate) is rotated by transporter 120 under control of processor 140in response to an observed angular position of substrate 10 asdetermined by processor 140 through viewing one or more fiducial markson substrate 10 (particularly fiducial marks 18) with a camera (notshown). This rotation will continue until substrate 10 has reached apredetermined angular relationship with respect to dispensing headsystem 210. In the case of a square or rectangular substrate, themounted substrate 10 will typically be rotated to align one edge (lengthor width) with the scan direction of head system 210 along axis 204.

Head system 210 may contain one or more (for example, two) heads 210 a,210 b mounted on the same head retainer 208. Each such head may be of atype commonly used in an ink jet type of printer and may, for example,have one hundred fifty drop dispensing orifices in each of two parallelrows, six chambers for holding polynucleotide solution communicatingwith the three hundred orifices, and three hundred ejectors which arepositioned in the chambers opposite a corresponding orifice. Eachejector is in the form of an electrical resistor operating as a heatingelement under control of processor 140 (although piezoelectric elementscould be used instead). Each orifice with its associated ejector andportion of the chamber, defines a corresponding pulse jet with theorifice acting as a nozzle. Thus, there are three hundred pulse jets inthis configuration, although it will be appreciated that head system 210could, for example, have more or less pulse jets as desired (forexample, at least ten or at least one hundred pulse jets). In thismanner, application of a single electric pulse to an ejector causes adroplet to be dispensed from a corresponding orifice. In the foregoingconfiguration, typically about twenty orifices in each group of sixreservoirs (many of the orifices are unused and are plugged with glue),will be dispensing the same fluid. Thus, each “series” in such aconfiguration has twenty dispensers. Certain elements of each head canbe adapted from parts of a commercially available thermal inkjet printhead device available from Hewlett-Packard Co. as part no. HP51645A. Theforegoing head system 210 and other suitable dispensing head designs aredescribed in more detail in U.S. patent application entitled “A MULTIPLERESERVOIR INK JET DEVICE FOR THE FABRICATION OF BIOMOLECULAR ARRAYS”Ser. No. 09/150,507 filed Sep. 9, 1998, incorporated herein byreference. However, other head system configurations can be used.

As is well known in the ink jet print art, the amount of fluid that isexpelled in a single activation event of a pulse jet, can be controlledby changing one or more of a number of parameters, including the orificediameter, the orifice length (thickness of the orifice member at theorifice), the size of the deposition chamber, and the size of theheating element, among others. The amount of fluid that is expelledduring a single activation event is generally in the range about 0.1 to1000 pL, usually about 0.5 to 500 pL and more usually about 1.0 to 250pL. A typical velocity at which the fluid is expelled from the chamberis more than about 1 m/s, usually more than about 10 m/s, and may be asgreat as about 20 m/s or greater. As will be appreciated, if the orificeis in motion with respect to the receiving surface at the time anejector is activated, the actual site of deposition of the material willnot be the location that is at the moment of activation in aline-of-sight relation to the orifice, but will be a location that ispredictable for the given distances and velocities.

The sizes of the features can have widths (that is, diameter, for around spot) in the range from a minimum of about 10 μm to a maximum ofabout 1.0 cm. In embodiments where very small spot sizes or featuresizes are desired, material can be deposited according to the inventionin small spots whose width is in the range about 1.0 μm to 1.0 mm,usually about 5.0 μm to 500 μm, and more usually about 10 μm to 200 μm.Spot sizes can be adjusted as desired, by using one or a desired numberof pulses from a pulse jet to provide the desired final spot size.

Multiple pulse jets of head 210 can be simultaneously loaded throughorifices (sometimes referenced as “nozzles”) of the pulse jets at aloading station (not shown). The loading station may have multiplereceptacles to retain multiple different fluids such that the dispenserscan be simultaneously brought into contact with respective receptaclesfor loading the dispensers with the different fluids. Such a loadingstation may include a flexible microtitre plate as described in U.S.patent application “Method and Apparatus for Liquid Transfer”, Ser. No.09/183,604. The transport system can move head 210 to such a loadingstation, as required.

The apparatus optionally may optionally include a sensor in the form ofa camera 304, to monitor for errors (such as failure to dispensedroplets) by monitoring for drops dispensed onto substrate 10 whenrequired of a dispenser. Camera 304 communicates with processor 140, andshould have a resolution that provides a pixel size of about 1 to 100micrometers and more typically about 4 to 20 micrometers or even 1 to 5micrometers. Any suitable analog or digital image capture device(including a line by line scanner) can be used for such camera, althoughif an analog camera is used processor 140 should include a suitableanalog/digital converter. A detailed arrangement and use of such acamera to monitor for dispenser errors, is described in U.S. patentapplication Ser. No. 09/419,447 entitled “Biopolymer Array Inspection”by William D. Fisher. Particular observations techniques are described,for example, in co-pending U.S. patent application Ser. No. 09/302,898filed Apr. 30, 1999 by Caren et al., assigned to the same assignee asthe present application, incorporated herein by reference.Alternatively, the sensor can be a drop detector which detects anelectrical charge on a dispensed drop, in accordance with the apparatusand methods described in U.S. Ser. No. 09/558,532 entitled “ArrayFabrication with Drop Detection” filed by Christopher A. Schantz et al.Monitoring can occur during formation of an array and the informationused during fabrication of the remainder of that array or another array,or test-print patterns can be run before array fabrication.

A display 310, speaker 314, and operator input device 312, are furtherprovided. Operator input device 312 may, for example, be a keyboard,mouse, or the like. Processor 140 has access to a memory 141, andcontrols print head system 210 (specifically, the activation of theejectors therein), operation of the transport system, operation of eachjet in print head system 210, capture and evaluation of images from thecamera 304, and operation display 310 and speaker 314. Memory 141 may beany suitable device in which processor 140 can store and retrieve data,such as magnetic, optical, or solid state storage devices (includingmagnetic or optical disks or tape or RAM, or any other suitable device,either fixed or portable). Processor 140 may include a general purposedigital microprocessor suitably programmed from a computer readablemedium carrying necessary program code, to execute all of the functionsrequired of it as described below. It will be appreciated though, thatwhen a “processor” such as processor 140 is referenced throughout thisapplication, that such includes any hardware and/or software combinationwhich will perform the required functions. Suitable programming can beprovided remotely to processor 140, or previously saved in a computerprogram product such as memory 141 or some other portable or fixedcomputer readable storage medium using any of those devices mentionedbelow in connection with memory 141. For example, a magnetic or opticaldisk 324 may carry the programming, and can be read by disk reader 326.A cutter 340 is provided which under control of processor 140 canseparate substrate 10 by cutting it, into multiple equal sized substratesegments 10 a each carrying at least one array 12.

Operation of the apparatus of FIG. 5 in accordance with a method of thepresent invention, will now be described. First, it will be assumed thatmemory 141 holds a target drive pattern. This target drive pattern isthe instructions for driving the apparatus components as required toform the target array (which includes target locations and dimension foreach spot) on substrate 10 and includes, for example, movement commandsto transporters 60 and 100 as well as firing commands for each of thepulse jets in head system 210 co-ordinated with the movement of headsystem 210 and substrate 10, as well as instructions for whichpolynucleotide solution (or precursor) is to be loaded in each pulse jet(that is, the “loading pattern”). This target drive pattern is basedupon the target array pattern and can have either been input from anappropriate source (such as input device 312, a portable magnetic oroptical medium, or from a remote server, any of which communicate withprocessor 140), or may have been determined by processor 140 based uponan input target array pattern (using any of the appropriate sourcespreviously mentioned) and the previously known nominal operatingparameters of the apparatus. Further, it will be assumed that drops ofdifferent biomonomer or biopolymer containing fluids (or other fluids)have been placed at respective regions of a loading station (not shown).Operation of the following sequences are controlled by processor 140,following initial operator activation, unless a contrary indicationappears.

For any given substrate 10, the operation of the present method isbasically follows. A target drive pattern is determined (if not alreadyprovided) to obtain multiple target arrays, based on nominal operatingparameters and target polynucleotide array pattern. The apparatus isthen operated as follows: (a) load head system 210 with a first set ofpolynucleotide containing solutions or their precursors; (b) dispensedroplets from head system 210 onto substrate 10 using multiple passes asdescribed above; and (c) optionally reload head 210 at the loadingstation and repeat steps (a) and (b) as desired until drops of allrequired solutions have been dispensed onto substrate 10 so as to formthe multiple arrays 12.

A loading sequence for head system 210 is more completely described inco-pending patent applications “FABRICATING BIOPOLYMER ARRAYS”, by Carenet al., Ser. No. 09/302,922, and “PREPARATION OF BIOPOLYMER ARRAYS” byA. Schleifer et al., Ser. No. 09/302,899, both filed Apr. 30, 1999 andboth assigned to the same assignee as the present application, and thereferences cited therein, including the possibility of using a flexiblemicrotitre plate as mentioned above. Those references and all otherreferences cited in the present application, are incorporated into thisapplication by reference. Processor 140 can control pressure within headsystem 210 to load each polynucleotide solution into the chambers in thehead by drawing it through the orifices as described in one or more ofthe foregoing applications.

Substrate 10 is loaded onto substrate station 20 either manually by anoperator, or optionally by a suitable automated driver (not shown)controlled, for example, by processor 140.

The deposition sequence is then initiated to deposit the desired arraysof polynucleotide containing fluid droplets on the substrate accordingto the target pattern to form the arrays each with respective featurelocations and dimensions. As already mentioned, in this sequenceprocessor 140 will operate the apparatus according to the target drivepattern, by causing the transport system to position head system 210facing substrate station 20, and particularly the mounted substrate 10,and with head system 210 at an appropriate distance from substrate 10.Processor 140 then causes the transport system to scan head system 210across substrate 10 line by line (or in some other desired pattern),while co-ordinating activation of the ejectors in head system 210 so asto dispense droplets, using multiple passes as described above. Asalready mentioned, if necessary or desired, processor 140 can repeat theloading and multiple passes one or more times until head system 210 hasdispensed droplets to obtain the target arrays 12 on substrate 10. Thenumber of spots in any one array 12 can, for example, be at least ten,at least one hundred, at least one thousand, or even at least onehundred thousand.

At this point the droplet dispensing sequence is complete. Substrate 10can then be sent to cutter 340 and cut into separate segments 10 a, eachcarrying at least one array 12.

When a user receives an array made by an apparatus or method of thepresent invention, it will typically be exposed to a sample and thearray interrogated following exposure. Interrogation is usuallyaccomplished by a suitable scanner which can read the location andintensity of fluorescence at each feature of an array following exposureto a fluorescently labeled sample (such as a polynucleotide containingsample). For example, such a scanner may be similar to the GENEARRAYscanner available from Hewlett-Packard, Palo Alto, Calif. Results fromthe interrogation can be processed such as by rejecting a reading for afeature which is below a predetermined threshold and/or formingconclusions based on the pattern read from the array (such as whether ornot a particular target sequence may have been present in the sample).The results of the interrogation or processing can be forwarded (such asby communication) to a remote location if desired, for further use.

The present methods and apparatus may be used to deposit biopolymers orother chemical moieties on surfaces of any of a variety of differentsubstrates, including both flexible and rigid substrates. Preferredmaterials provide physical support for the deposited material and endurethe conditions of the deposition process and of any subsequent treatmentor handling or processing that may be encountered in the use of theparticular array. The array substrate may take any of a variety ofconfigurations ranging from simple to complex. Thus, the substrate couldhave generally planar form, as for example a slide or plateconfiguration, such as a rectangular or square or disc. In manyembodiments, the substrate will be shaped generally as a rectangularsolid, having a length in the range about 4 mm to 1 m, usually about 4mm to 600 mm, more usually about 4 mm to 400 mm; a width in the rangeabout 4 mm to 1 m, usually about 4 mm to 500 mm and more usually about 4mm to 400 mm; and a thickness in the range about 0.01 mm to 5.0 mm,usually from about 0.1 mm to 2 mm and more usually from about 0.2 to 1mm. However, larger substrates can be used, particularly when such arecut after fabrication into smaller size substrates carrying a smallertotal number of arrays 12.

In the present invention, any of a variety of geometries of arrays on asubstrate 10 may be fabricated other than the rectilinear rows andcolumns of arrays 12 of FIG. 1. For example, arrays 12 can be arrangedin a sequence of curvilinear rows across the substrate surface (forexample, a sequence of concentric circles or semi-circles of spots), andthe like. Similarly, the pattern of features 16 may be varied from therectilinear rows and columns of spots in FIG. 2 to include, for example,a sequence of curvilinear rows across the substrate surface (forexample, a sequence of concentric circles or semi-circles of spots), andthe like. In such cases, the arrangement of dispensers in head system210 may be altered accordingly. The configuration of the arrays andtheir features may be selected according to manufacturing, handling, anduse considerations.

The substrates may be fabricated from any of a variety of materials. Incertain embodiments, such as for example where production of bindingpair arrays for use in research and related applications is desired, thematerials from which the substrate may be fabricated should ideallyexhibit a low level of non-specific binding during hybridization events.In many situations, it will also be preferable to employ a material thatis transparent to visible and/or UV light. For flexible substrates,materials of interest include: nylon, both modified and unmodified,nitrocellulose, polypropylene, and the like, where a nylon membrane, aswell as derivatives thereof, may be particularly useful in thisembodiment. For rigid substrates, specific materials of interestinclude: glass; plastics (for example, polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like).

The substrate surface onto which the polynucleotide compositions orother moieties is deposited may be smooth or substantially planar, orhave irregularities, such as depressions or elevations. The surface maybe modified with one or more different layers of compounds that serve tomodify the properties of the surface in a desirable manner. Suchmodification layers, when present, will generally range in thicknessfrom a monomolecular thickness to about 1 mm, usually from amonomolecular thickness to about 0.1 mm and more usually from amonomolecular thickness to about 0.001 mm. Modification layers ofinterest include: inorganic and organic layers such as metals, metaloxides, polymers, small organic molecules and the like. Polymeric layersof interest include layers of: peptides, proteins, polynucleic acids ormimetics thereof (for example, peptide nucleic acids and the like);polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated).

Various further modifications to the particular embodiments describedabove are, of course, possible. Accordingly, the present invention isnot limited to the particular embodiments described in detail above.

1. An apparatus for fabricating multiple arrays of biopolymers arrangedsuccessively in a first direction on a substrate and each havingmultiple feature sets arranged successively in the first directionwithin the array, the apparatus comprising: (a) a head system withmultiple successive sets of dispensers wherein the head system is loadedwith biopolymers or biomonomers; (b) a transport system to advance thehead system in the first direction with respect to a substrate; and (c)a processor communicating with the head system and transport system toadvance the head system in the first direction over the substrate whiledispensing drop sets for each array, from dispenser sets in an order thereverse of that from which the dispenser sets pass over a given locationon the substrate as the head system advances in the first direction,with each dispenser set depositing a drop set at a distance ahead of adrop set deposited by a preceding dispenser set which is less than thedistance to the successive drop dispenser set which deposits the nextdrop set, so as to form the arrays.
 2. An apparatus according to claim 1wherein the distance between adjacent feature sets within the arrays isless than the distance between adjacent dispenser sets.
 3. An apparatusaccording to claim 1 wherein the advancing and dispensing comprises: (a)while the head system is in one position in the first direction,depositing drop sets from different dispenser sets for feature sets ofdifferent positions within multiple arrays; (b) advancing the headsystem in the first direction to a next position; and (c) repeatingsteps (a) and (b) for successive feature sets within the arrays usingfor each of those feature sets during a cycle, a corresponding dispenserset which deposited at a same feature set position of a previous arrayin step (a) during a previous cycle.
 4. An apparatus according to claim1 wherein the feature sets are rows of features, the sets of dispensersare rows of dispensers, and the dispensed drop sets are rows of drops.5. An apparatus according to claim 1 wherein the dispensers are pulsejets.
 6. An apparatus according to claim 1 wherein the head system isloaded with biomonomers.
 7. An apparatus according to claim 1 whereinthe head system is loaded with polynucleotides, peptides, nucleotides,or amino acids.
 8. An apparatus according to claim 1 wherein the headsystem is loaded with polynucleotides or peptides.
 9. An apparatusaccording to claim 8 wherein the head system is loaded withpolynucleotides.
 10. An apparatus according to claim 1 wherein the headsystem is loaded with nucleotides or amino acids.
 11. An apparatusaccording to claim 10 wherein the head system is loaded withnucleotides.
 12. A computer program product comprising a computerreadable storage medium having a computer program stored thereon for usewith an apparatus for fabricating multiple arrays arranged successivelyin a first direction on a substrate and each having multiple featuresets arranged successively in the first direction within the array,which apparatus has: (i) a head system with multiple successive sets ofdispensers wherein the head system is loaded with biopolymers orbiomonomers; (ii) a transport system to advance the head system in afirst direction with respect to a substrate; and (iii) a processorcommunicating with the head system and transport system; the computerprogram product, when loaded into the processor, performs the steps of:advancing the head system in the first direction over the substratewhile dispensing drop sets for each array from dispenser sets in anorder the reverse of that from which the dispenser sets pass over agiven location on the substrate as the head system advances in the firstdirection, with each dispenser set depositing a drop set at a distanceahead of a drop set deposited by a preceding dispenser set which is lessthan the distance to the successive drop dispenser set which depositsthe next drop set, so as to form the arrays.
 13. A computer programproduct according to claim 12 wherein the distance between adjacentfeature sets within the arrays is less than the distance betweenadjacent dispenser sets.
 14. A computer program product according toclaim 12 wherein the advancing and dispensing comprises: (a) while thehead system is in one position in the first direction, depositing dropsets from different dispenser sets for feature sets of differentpositions within multiple arrays; (b) advancing the head system in thefirst direction to a next position; and (c) repeating steps (a) and (b)for successive feature sets within the arrays using for each of thosefeature sets during a cycle, a corresponding dispenser set whichdeposited at a same feature set position of a previous array in step (a)during a previous cycle.